The transcriptional regulation of a putative hemicellulose gene, PtrPARVUS2 in poplar

The plant cell wall serves as a critical interface between the plant and its environment, offering protection against various stresses and contributing to biomass production. Hemicellulose is one of the major components of the cell wall, and understanding the transcriptional regulation of its production is essential to fully understanding cell wall formation. This study explores the regulatory mechanisms underlying one of the genes involved in hemicellulose biosynthesis, PtrPARVUS2. Six transcription factors (TFs) were identified from a xylem-biased library to negatively regulate PtrPARVUS2 expression. These TFs, belonging to diverse TF families, were confirmed to bind to specific cis-elements in the PtrPARVUS2 promoter region, as validated by Yeast One-Hybrid (Y1H) assays, transient expression analysis, and Chromatin Immunoprecipitation sequencing (ChIP-seq) assays. Furthermore, motif analysis identified putative cis-regulatory elements bound by these TFs, shedding light on the transcriptional regulation of SCW biosynthesis genes. Notably, several TFs targeted genes encoding uridine diphosphate glycosyltransferases (UGTs), crucial enzymes involved in hemicellulose glycosylation. Phylogenetic analysis of UGTs regulated by these TFs highlighted their diverse roles in modulating hemicellulose synthesis. Overall, this study identifies a set of TFs that regulate PARVUS2 in poplar, providing insights into the intricate coordination of TFs and PtrPARVUS2 in SCW formation. Understanding these regulatory mechanisms enhances our ability to engineer plant biomass for tailored applications, including biofuel production and bioproduct development.

The plant cell wall is a key structure in mitigating the interaction between the plant and its environment.The physical structure of the plant cell wall reduces insect predation, plays important roles in intercellular communication, and even prevents unwanted molecules from entering the cell [1][2][3][4] .At the same time, the cell wall accounts for the majority of the organic carbon in the world, making it important from the perspective of biomass for fuels and other products 5 .
The secondary cell wall (SCW), located between the plasma membrane and the primary cell wall, is one of the factors that allows trees to grow to such great heights 6 .The SCW is predominantly comprised of cellulose, hemicelluloses, and lignin.Over the past 50 years, much research has been carried out to elucidate the biosynthetic pathways involved in the production of cellulose and lignin 7,8 .However, until more recently hemicellulose has not been as well studied.Hemicellulose plays an important role in plant biomass 9 and increasing or altering the content of hemicellulose has been identified as a potential way to raise the value of plant biomass for biofuels and other bioproducts.
In most vascular plants, such as poplar and Arabidopsis, the primary hemicellulose in the SCW is glucuronoxylan (xylan), with glucomannan and galactoglucomannan making up small proportions 10 .While cellulose is synthesized at the plasma membrane via large protein complexes, glucuronoxylan, a polymer of β-(1,4)-linked xylose with various side chains, is produced in the Golgi apparatus and transferred to the plasma membrane 11,12 .
In Arabidopsis, the IRX8 and PARVUS genes are vital for synthesizing the tetrasaccharide end sequence putatively involved in xylan biosynthesis 13 .This reducing end sequence (RES) is composed of D-Xylose, D-Glucuronic Acids, L-Rhamnose, D-Galactose, and L-Arabinose (furanose) 12 .While IRX8 operates in the Golgi, PARVUS, a member of the GT8 family, functions in the endoplasmic reticulum (ER), initiating an enzymatic stage preceding IRX8 13 .The different subcellular localizations indicate that IRX8 and PARVUS perform distinct functions in xylan biosynthesis.In Eucalyptus, expression of the PtrPARVUS2 ortholog is reduced in tension wood, indicating a putative role in stem formation 14 .In Populus trichocarpa, PtrPARVUS2 (Potri.002G132900)exhibits a high degree of stem tissue specificity, with heightened expression in the 7th-8th stem internodes, while its transcript

Generation of plasmid constructs
The 2100 bp region upstream of the start codon of PtrPARVUS2 was synthesized (Genewiz, NJ, USA).The unique restriction sites (XbaI and SbfI) were introduced to 5' and 3' of the promoter of PtrPARVUS2 (PtrPARVUS2 pro ) using the following primers: PARVUS-Fwd-XbaI and PARVUS-Rvs-SbfI (Supplemental Table S1).PtrPARVUS2 pro was cloned into the pGEM-T Easy vector (Promega, USA).The ligated vector was confirmed by PCR and Sanger sequencing.Using the pGFPGUSPlus binary expression vector 19 that harbors EGFP gene driven by the Cauliflower Mosaic Virus 35S (CaMV35S) promoter (RRID: Addgene_64401), we replaced the CaMV35S promoter with the PtrPARVUS2 pro by digesting both with XbaI and SbfI followed by T4 ligation (Promega, USA).The pGFPGUSPlus-PtrPARVUS2 pro ::EGFP vector was confirmed by PCR and Sanger sequencing and transformed into Agrobacterium tumefaciens (GV3101).

Plant materials
Populus tremula × alba (717-1B4; INRA France) was cultured in Woody Plant Medium (WPM) at 25 °C under a cycle of 16-h light (50 μmol m −2 s −1 )/8-h dark 22 .Transgenic hybrid poplar carrying the PtrPARVUS2 pro ::EGFP were generated through Agrobacterium tumefaciens-mediated transformation as previously described 23 .Following selection on media with antibiotics, PCR and SDS-PAGE were used to confirm transgenic lines.The transgenic plants were maintained in WPM for further analysis.Experiments on plants/plant parts must confirm that the use of plants in the present study complies with international, national, and/or institutional guidelines.

Transient expression assays
The pMDC84-CaMV35S::TF-GFP and positive control 35S::GFP binary vectors were transformed into Agrobacterium tumefaciens strain GV3101 as previously described 26 .The whole plants of Populus tremula × alba (717-1B4; INRA France) from WPM were soaked in the Agrobacterium solution containing 0.015% Silwet L-77 (Lehle Seeds, USA) and then Agrobacterium infiltration was performed by applying vacuum three times for three minutes.The vacuumed poplar was subsequently put on paper towels to remove excess infiltration medium and moved back to cubes at 25 °C under a cycle of 16 h light and 8 h dark for 7 days.RT-qPCR and western blot were applied for detecting TF::GFP fusion protein expression.The CaMV35S::TF and negative control empty binary vectors were transformed into Agrobacterium tumefaciens strain GV3101, the same agroinfiltration method was applied in PtrPARVUS2 pro ::EGFP stable transgenics.

RNA isolation and quantitative real-time PCR analysis
The tissue culture leaves were harvested after transient transformation and stored at − 80 °C until used.The tissues were ground into fine powder under liquid nitrogen.The total RNA was extracted by Tri-Xtract following manufacturer's instruction (G-Biosciences, USA), and 1 ug RNA treated with RQ1 RNase-Free DNase (Promega, WI, USA).DNase-treated RNA was reverse-transcribed to cDNA by High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, MA, USA).The expression of GFP was measured using qPCR.Samples were run in triplicate using SYBR ® Green master mix (BIO-RAD, CA, USA) on a CFX Connect™ Real-Time PCR Detection System (BIO-RAD, CA, USA).Poplar UBIQUITIN 11 (UBQ11, Potri.017G036800) and Elongation Factor 1-β 2 (EF1β, Potri.015G094200) were used as reference genes 27 .The following primer sets were used for qPCR: UBQ11-Fwd, UBQ11-Rvs, EF1β-Fwd, EF1β-Rvs, GFP-Fwd, GFP-Rvs (Supplemental Table S1).

SDS-PAGE and western blot analyses
Total protein was extracted from tissue culture leaves after transient transformation using a protein extraction buffer containing 150 mM Phosphate Buffer, 10 mM EDTA, 25 mM Sodium Metabisulphite, 100 mg/L PVP, 0.1% Triton X-100, 0.1% SDS and 0.07% β-mercaptoethanol (Côté et al., 2003).Briefly, 1 ml extraction buffer was added to 50 mg ground tissues and held on ice for 10 min.All of samples were centrifuged at 13,000 rpm for 10 min at 4 °C.800 μl of supernatant was removed to a fresh 1.5 ml tube and kept on ice for immediate use or snap frozen in liquid nitrogen and stored at − 80 °C.The total proteins were separated by 10% SDS-PAGE and stained by Coomassie Blue.The separated proteins were transferred onto a PVDF membrane as previously described 28 and detected by Mouse anti GFP-Tag monoclonal antibody (ABclonal, USA) and HRP Goat Anti-Mouse IgG (H + L) as the secondary antibody (ABclonal, USA).SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, USA) was used to detect HRP.Finally, chemiluminescence was captured by ChemiDoc MP Imaging System (Bio-Rad, USA) and analyzed by ImageJ.

Chromatin Immunoprecipitation sequencing (ChIP-seq) assays
After transfection and successful validation of GFP signal, 8 g of leaf tissue was harvested and immediately cross-linked with 1% formaldehyde under vacuum for 15 min at 25 °C.The cross-linking was stopped by adding glycine to the final concentration of 0.125 M for 5 min under vacuum.The cross-linked samples were washed three times with double distilled water.The ChIP assay based on GFP Monoclonal Antibody (Invitrogen, USA) was performed as described previously 29,30 .Chromatin samples without GFP antibody immunoprecipitation were used as the control.DNA was purified and the library was prepared by NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, USA).The DNA libraries were sequenced by Illumina NextSeq 2000 next-generation sequencing system from SUNYMAC (SUNY Molecular Analysis Core).
For ChIP-Seq data processing, the paired-end reads were trimmed by Trimmomatic 31 .After trimming the adapter sequence, clean reads were mapped to the Populus tremula × alba genome (https:// urgi.versa illes.inra.fr/ Speci es/ Forest-trees/ Popul us/ Clone-INRA-717-1B4) by Bowtie2 with default parameters 32 .Mapped reads with low mapping quality (MAPQ < 30) or multiple duplicates removed by SAMtools 33 .Enriched peaks were identified by MACS 34 .We defined the region of a target gene as the range from 2.5 kb upstream of transcription start site (TSS) to transcription terminal site (TTS).The target genes of each peak were annotated by annotate Peak function in Chipseeker R package 35 .plotProfile and plotHeatmap in deepTools were used for peak visualization 36 .To identify DNA motifs, the peak files were searched for enriched DNA motifs using the MEME 37 .Peak files were visualized by Integrated Genome Browser 38 .

TFs bind to the promoter of PtrPARVUS2
To determine whether the 42 TFs from the xylem biased library interact with the promoter of PtrPARVUS2, the 2.1 kb promoter was cloned in pMW#2 and pMW#3 vectors, which harbor HIS3 and LacZ report genes for Y1H assays.Of the transformed yeast cells with two different reporters, only PtrC2H2ZF1, PtrC2H2ZF2, PtrARF5a, PtrBLH, PtrNAC127 and PtrCORONA grew on SC-His/-Ura/-Trp medium with 0 mM and 80 mM 3-AT (Fig. 1, Supplemental Fig. S1).At the same time, X-Gal was active and turned blue in these transformed yeast cells.These Vol:.( 1234567890

TFs localize to the nucleus
To investigate the subcellular localization of six TFs in vivo, we transformed CaMV35S::TF-GFP into onion epidermal cells by Agrobacterium-mediated transient transformation, using CaMV35S empty vector and CaMV35S::GFP as negative and positive controls.CaMV35S::PtrC2H2ZF1-GFP, CaMV35S::PtrC2H2ZF2-GFP, CaMV35S::PtrARF5a-GFP, CaMV35S::PtrNAC127-GFP and CaMV35S::PtrCORONA-GFP expression was observed in the nuclei, which were also stained by DAPI (Fig. 2).CaMV35S::PtrBLH-GFP was expressed in both the cytoplasm and nucleus.By contrast, CaMV35S::GFP signals were uniformly distributed throughout the cell, and there was no GFP signal in empty vector transformed cell, indicating that PtrC2H2ZF1, PtrC2H2ZF2, PtrARF5a, PtrNAC127 and PtrCORONA are nuclear proteins.The PtrBLH expression pattern indicated that it might have another function besides as a transcription factor.

TFs function as transcriptional repressors of PtrPARVUS2
Previous transcriptome analysis showed that PtrBLH and PtrNAC127 had the opposite expression pattern in the stem as PtrPARVUS2, which had expression in 1st-4th stem internodes, but not in 7th-8th stem internodes 15 indicated that PtrBLH and PtrNAC127 could be repressors of PtrPARVUS2.To gain insight into the transcriptional role of these six TFs, the TFs were transiently expressed in leaf tissues of PtrPARVUS2 pro ::EGFP transgenics through agroinfiltration (Fig. 3a).EGFP transcriptional levels were detected by RT-qPCR, using empty vector as the control.The results show that EGFP transcriptional levels were decreased after overexpressing PtrC2H2ZF1, PtrC2H2ZF2, PtrARF5a, PtrNAC127, PtrBLH and PtrCORONA (Fig. 3b), indicating that these six TFs can negatively regulate PtrPARVUS2.

TFs target genes identified by ChIP-Seq
To identify what additional genes can be targeted by these six TFs, ChIP-Seq was performed by immunoprecipitating TF-GFP fusion protein and the cross-linked DNA.~ 15 million raw reads per sample were obtained from sequencing.PtrC2H2ZF1, PtrC2H2ZF2, PtrARF5a, PtrBLH, PtrNAC127 and PtrCORONA bind predominantly to promoter regions, particularly the regions around the transcriptional start site (TSS) and that have a moderate enrichment at transcribed gene body regions (Fig. 4).A total of 575 putative binding sites were identified in the PtrC2H2ZF1 sample, with the majority (54%) located in the promoter, 42% in distal intergenic regions, and 4% in other areas such as introns and exons (Fig. 5a).Similarly, in the PtrC2H2ZF2 sample, a total of 607 putative binding sites were found, with 60% in the promoter, 37% in distal intergenic regions, and 3% in other regions (Fig. 5b).In the PtrARF5a sample, 5397 putative binding sites were identified, with 57% in the promoter, 34% in distal intergenic regions, and 9% in other regions (Fig. 5c).In the PtrBLH sample, 1022 putative binding sites were identified, with 66% in the promoter, 32% in distal intergenic regions, and 2% in other regions (Fig. 5d).For PtrNAC127, a total of 682 putative binding sites were found, with 63% in the promoter, 34% in distal intergenic regions, and 3% in other regions (Fig. 5e).Lastly, in the PtrCORONA sample, 1106 putative binding sites were found, with 57% in the promoter, 40% in distal intergenic regions, and 3% in other regions like introns and exons (Fig. 5f).Approximately 75% of the binding sites within the promoter regions targeted by these six TFs were concentrated within the 2 kb region, signifying their active involvement in the transcription binding process.The full list of the genes targeted by the six TFs is in Supplemental Table S3-S8.

Binding motif analysis reveals cis-regulatory elements for these six TFs
To explore the binding motifs from PtrC2H2ZF1, PtrC2H2ZF2, PtrARF5a, PtrBLH, PtrNAC127 and PtrCO-RONA, 2 kb flanking sequences around all peaks were analyzed by the motif discovery tool MEME described in Methods section.The highest score motifs from each TFs were showed in Fig. 5a-f S2).These results indicate that the genes with these motifs in promoter regions could be regulated by these six TFs directly.

Discussion
The present research aimed to identify TFs from a xylem biased library for their ability to regulate PtrPARVUS2 expression by targeting specific cis-elements in the promoter region.The investigation into the regulatory network controlling SCW development in plants, with a focus on the TF-mediated regulation of the PtrPARVUS2 gene, provides valuable insights into the molecular mechanisms underlying cell wall formation.We have showed that six TFs can bind to PtrPARVUS2 promoter region to activate the downstream HIS3 and LacZ reporter genes in Y1H assays, and that these six TFs are negative transcriptional regulators of PtrPARVUS2.These six TFs from five different families were reported to have multiple functions in cell development and different stress responses, but the function in SCW biosynthesis has not been fully studied.PtrC2H2ZF1 (Potri.010G209400)and PtrC2H2ZF2 (Potri.014G066200)are from C2H2 zinc finger (C2H2-ZF) TF family.The first discovery of zinc-binding domains occurred in the protein TFIIIA from Xenopus oocytes.These domains play a crucial role in binding to IIA and DNA, utilizing cysteines and histidines within the enriched folding domain centered around a zinc ion 39 .The majority of classical Cys2His2 (C2H2) zinc fingers function as transcription factors, contributing to DNA binding and transcriptional regulation through the β-β-α framework of a C2H2 zinc finger module (Supplemental Fig. S5) 40 .The functions of C2H2 zinc fingers in growth regulation, stress response and epigenetic have been reported in plants [41][42][43] .However, the role of C2H2 zinc fingers in the SCW formation in poplar remains poorly understood.
PtrARF5a (Potri.002G024700) is from Auxin Response Factor family.39 ARF genes have been identified in P. trichocarpa 44 .Each ARF protein features a conserved DNA-binding domain (DBD) at its N-terminus, followed by a middle region (MR) and a C-terminal PB1 domain 45 .The N-terminal DBD is highly conserved among ARFs, however, the MR exhibits variability depending on whether it is associated with activating or repressing functions.When the middle region (MR) is characterized by an abundance of glutamines, it functions as an activator.Conversely, a middle region rich in proline, serine, and threonine imparts repressor activity 44 .But the mechanism of activation and repression is still waiting to be elucidated.The function of ARFs in the cell wall is being revealed, as ARFs can regulate the cell elongation, targeting genes involved in cellulose and pectin biosynthesis in the primary cell wall 46 .Following treatment with IAA (Indole-3-Acetic Acid) in Arabidopsis, AtPARVUS (AT1G19300), the ortholog of PtrPARVUS2, exhibits upregulation within 120 minutes 47 .It indicated that auxin-related receptors could be involved in PtrPARVUS2 transcriptional regulation.
PtrBLH (Potri.010G197300) is BEL1-Like Homeodomain protein from homeobox protein transcription factor family.Three-amino-acid-loop-extensions and a conserved proline-tyrosine-proline amino acid sequence within the homeodomain are two key characteristics of BLH protein 48 .The domains located at N-terminus and C-terminus are conserved, homeodomain serves as DNA-binding domain at its N-terminus, POX domain is conserved at C-terminus, involving in homodimer or heterodimer formation 49,50 .Eighteen BLH genes have been identified in the poplar genome 51 , but the role of each of these genes has not yet been elucidated.PtrBLH6a has been identified as a negative regulator of genes involved in the lignin biosynthesis pathway, the overexpression mutants did not exhibit any alterations in lignin composition (S/G ratio) and content 52 .In contrast, blh6 mutants in cotton and camellia show a reduction in lignin content 53,54 .In addition to a role in regulating SCW formation, BLHs are also reported to have vital roles in salt stress, heat stress and other pathogen infections 51,55 .
PtrNAC127 (Potri.018G068700) is from NAM/ATAF1/CUC2 (NAC) transcription factor family, the largest plant-specific TF family involved in biotic and abiotic stress response and plant development [56][57][58] .The NAC proteins encompass a C-terminal transcription regulation region (TRR) that is highly variable, featuring a substantial content of low-complexity amino acid repeats, and N-terminal with a 150-amino-acid DNA-binding domain (DBD) is conserved in NAC TFs 59 .The TRR acts as both activator and/or repressor to downstream target genes together with the NAC domain 60 .The NAC domain is present in all NAC proteins, with the most conserved consensus sequences being D-D/E-L-I/V, E-W-Y-F-F, G-Y-W-K, and M-H-E-Y 61 .Nuclear localization signals (NLSs) within the NAC domain have been identified, indicating their role in DNA-binding functions 62 .Overexpression of PtrWND1B, PtrWND2B and PtrWND6B, other members of Wood-associated NAC Domain (WND) transcription factor family, results in the increased expression of IRX8, IRX9, FRA8 and GT43B involved in xylan biosynthesis in hemicellulose [63][64][65] .PtrWND4A and PtrWND4B, orthologs of AtVND4/5 with the same the NAC domain as PtrNAC127 in poplar, have different expression patterns; PtrWND4A cannot be expressed in the petiole, and PtrWND4B has an extremely low expression in the leaf 66 .When AtSND2 was overexpressed in Arabidopsis, xylose content did not show a significant change, but PtrNAC154 which is the ortholog of AtSND2 in poplar can decrease xylose content significantly after overexpression in poplar 67,68 .The ortholog of PtrNAC127 in Arabidopsis is AtSND4, which can bind to the 19-bp consensus sequence named SNBEs (secondary wall NAC binding elements) to regulate downstream TFs involved in SCW formation 69,70 , and the evidence that PtrNAC127 can regulate the SCW biosynthesis genes was provided in this study.
PtrCORONA (Potri.001G188800) is a member of Class III Homeodomain-Leucine Zipper (HD ZIP III) transcription factor family.This family is grouped in three clades: REVOLUTA (REV), PHABULOSA/PHAVO-LUTA (PHB/PHV), CORONA/AtHB15 (CNA) 71 .HD-ZIP III proteins contain a unique START domain located at N-terminal for steroid binding and a C-terminal MEKHLA (Met-Glu-Lys-His-Leu-Ala) domain, which is responsive to various chemical and physical stimuli 72 .The HD ZIP III genes play a crucial role in governing plant development, including meristem development, leaf polarity, and vascular development in both stems and roots [73][74][75][76][77] .The recent studies indicate that post-transcriptional regulation of HD ZIP III genes is associated with various abiotic stresses in a number of species [78][79][80][81] .In poplar, PtrREV and PtrCORONA have been reported to regulate vascular cambium development and cell differentiation during secondary growth 73,82 .
Furthermore, hierarchical regulatory networks have been previously reported for transcription factors involved in plant stress responses and secondary metabolism 83,84 6), highlighting the complexity of transcriptional regulation in SCW biosynthesis.
Among the target genes of PtrARF5a are PtrGT8E, PtrPARVUS14, PtrGUX1A, PtrCel9A1 and PtrCesA8B, which are key enzymes related to cell wall formation.PtrGT8E and PtrPARVUS14 are close homologs of PtrPAR-VUS2 and have important roles in xylan backbone biosynthesis 85 .PtrGUX1A plays a role in acetyl modification, demonstrating a proclivity for even modification along the backbone 86 .In addition to these xylan biosynthesis and modification genes, cellulose biosynthesis genes are also the targets of PtrARF5a including PtrCel9A1 and PtrCesA8B.Suppression of either PtrCel9A1 or PtrCesA8B in poplar can lead to defects in xylem cells and significant cellulose reduction in SCW 87,88 .These findings showed that PtrARF5a has a multiple regulatory function role in SCW formation, and auxin could be a key SCW formation regulator.
GO analysis reveals that numerous binding sites are located within the promoters of genes linked to UGT activity (Supplemental Fig. S3).UGTs are known to catalyze the transfer of sugar moieties from uridine diphosphate (UDP) sugar donors to various acceptor molecules, leading to the formation of glycosidic bonds 89 .In the context of SCW biosynthesis, UDP-glycosyltransferases are involved in the glycosylation of hemicelluloses like xylan, influencing its structure and function 90 .The substrate specificity of UGTs determines the precise glycosylation patterns of hemicelluloses within SCW.In poplar, 191 putative UGTs have been identified 91 , but their regulatory mechanisms have not been elucidated yet.The eight putative UGTs regulated by all six TFs can be grouped into three clades using promoter phylogenetic analysis (Supplemental Fig. S4).According to previous research, genes in clades I and III are associated with two different environmental stresses.Potri.015G027700,classified in clade I, has been significantly downregulated under heat shock in poplar 92 and Potri.006G022500 and Potri.017G052400from clade III are activated under drought stress 91,93,94 .Understanding the regulation of UGTs in different plant species can provide valuable insights into the variability of cell wall structures and properties.The intricate network of TFs involved in SCW biosynthesis interacts with UGTs to coordinate the expression of genes responsible for hemicellulose synthesis.This interplay ensures the precise spatiotemporal regulation of cell wall components.In this study, six different TFs have shown the abilities to regulate PtrPARVUS2 from GT8 family through Y1H, ChIP-seq and transient expression (Figs. 1, 3 and 5).The identification of TFs associated with UGTs will contribute to a more comprehensive view of the regulatory mechanisms governing SCW formation.

Conclusion
Our results show that PtrC2H2ZF1, PtrC2H2ZF2, PtrARF5a, PtrNAC127, PtrBLH and PtrCORONA can bind to PtrPARVUS2 promoter in vivo and, apart from PtrBLH are localized in the nuclei.These six TFs suppress PtrPARVUS2 transcription by targeting different cis-elements in the promoter region directly or indirectly.These results provide valuable insights into the transcriptional regulatory mechanisms of PtrPARVUS2 in plant development.Further understanding the transcriptional network regulating PtrPARVUS2 will lead to novel strategies for engineering xylan in hemicellulose.

Figure 1 .
Figure 1.Y1H analysis of interactions between six transcription factors and the PtrPARVUS2 promoter.pMW#2-PtrPARVUS2pro and pMW#3-PtrPARVUS2 pro with HIS3 and LacZ reporter genes were used for the bait, six TFs fused with activation domain were used as prey.The empty pDEST-AD vector was used as the negative control, the positive yeast stain was a gift from Sarah Hall (Syracuse University).
, the motif HRGT CAA CB (E-value = 4.4E − 22) demonstrated statistical significance as the predominant binding motif in PtrC2H2ZF1

Figure 3 .
Figure 3. (a) Diagrams of the effector and reporter vectors used in transient expression assays.(b) Analysis of EGFP in leaf tissues after transient expression by RT-qPCR.Expression values are average of three biological replicates ± standard error of the mean (SEM), normalized to the reference UBQ11 and EF1β, respectively.The error bars represent standard deviations from three biological replicates.****P < 0.0001 based on t-test.

Figure 4 .
Figure 4.The distribution of ChIP signals in the gene body and flanking regions.TSS, transcription start site; TTS, transcription termination site; 2.5, 2.5 kb downstream of the TTS; -2.5, 2.5 kb upstream of the TSS.

Figure 5 .
Figure 5. Distribution of peaks in different genomic regions and correspondent motifs.In each pattern from a to f, pie chart showing the distribution of ChIP peaks in genic and distal intergenic regions in each TF, the motif showed as sequence LOGO was significantly enriched in TF-binding regions in each TF.

Figure 6 .
Figure 6.A proposed regulatory network among multiple TFs.(a) RT-qPCR was performed after transient expression of six TFs.The y-axis illustrated the overexpressed 6 TFs, while the x-axis depicted the detected expression levels of 6 TFs.Expression values are the average of three biological replicates ± standard error of the mean (SEM), normalized to the reference UBQ11 and EF1β, respectively.The scale bar showed the range of relative expression levels.(b) Model of regulation network among 6 TFs in poplar based on RT-qPCR and ChIPseq assay.